Department of Molecular, Cellular, and Developmental Biology, Yale University, New Haven, Connecticut c

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1 The Plant Cell, Vol. 22: , January 2010, ã 2010 American Society of Plant Biologists Arabidopsis CULLIN4-Damaged DNA Binding Protein 1 Interacts with CONSTITUTIVELY PHOTOMORPHOGENIC1-SUPPRESSOR OF PHYA Complexes to Regulate Photomorphogenesis and Flowering Time C W Haodong Chen, a,b,c,1 Xi Huang, b,c,d,1 Giuliana Gusmaroli, b William Terzaghi, b,e On Sun Lau, b Yuki Yanagawa, b,2 Yu Zhang, a,c Jigang Li, a,b Jae-Hoon Lee, b Danmeng Zhu, a,b,c and Xing Wang Deng a,b,c,3 a Peking-Yale Joint Center for Plant Molecular Genetics and Agro-biotechnology, National Laboratory of Protein Engineering and Plant Genetic Engineering, College of Life Sciences, Peking University, Beijing , China b Department of Molecular, Cellular, and Developmental Biology, Yale University, New Haven, Connecticut c National Institute of Biological Sciences, Beijing , China d College of Life Sciences, Beijing Normal University, Beijing, , China e Department of Biology, Wilkes University, Wilkes-Barre, Pennsylvania CONSTITUTIVELY PHOTOMORPHOGENIC1 (COP1) possesses E3 ligase activity and promotes degradation of key factors involved in the light regulation of plant development. The finding that CULLIN4 (CUL4)-Damaged DNA Binding Protein1 (DDB1) interacts with DDB1 binding WD40 (DWD) proteins to act as E3 ligases implied that CUL4-DDB1 may associate with COP1-SUPPRESSOR OF PHYA (SPA) protein complexes, since COP1 and SPAs are DWD proteins. Here, we demonstrate that CUL4-DDB1 physically associates with COP1-SPA complexes in vitro and in vivo, likely via direct interaction of DDB1 with COP1 and SPAs. The interactions between DDB1 and COP1, SPA1, and SPA3 were disrupted by mutations in the WDXR motifs of MBP-COP1, His-SPA1, and His-SPA3. CUL4 cosuppression mutants enhanced weak cop1 photomorphogenesis and flowered early under short days. Early flowering of short day grown cul4 mutants correlated with increased FLOWERING LOCUS T transcript levels, whereas CONSTANS transcript levels were not altered. De-etiolated1 and COP1 can bind DDB1 and may work with CUL4-DDB1 in distinct complexes, but they mediate photomorphogenesis in concert. Thus, a series of CUL4-DDB1-COP1-SPA E3 ligase complexes may mediate the repression of photomorphogenesis and, possibly, of flowering time. INTRODUCTION Light is an essential signal for plant development and is involved in diverse processes throughout the life of a plant, ranging from germination and seedling photomorphogenesis to flowering. Light-grown seedlings exhibit short hypocotyls and opened and expanded cotyledons, while dark-grown seedlings show long hypocotyls, closed cotyledons, and apical hooks. The two developmental strategies adopted in light and dark conditions were named photomorphogenesis and skotomorphogenesis, respectively (von Arnim and Deng, 1996). At the adult stage, the transition from vegetative to reproductive development in plants 1 These authors contributed equally to this work. 2 Current address: Plant Science Education Unit, Graduate School of Biological Sciences, Nara Institute of Science and Technology, Takayama, Ikoma-shi, Nara , Japan. 3 Address correspondence to xingwang.deng@yale.edu. The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors ( is: Xing Wang Deng (xingwang.deng@yale.edu). C Some figures in this article are displayed in color online but in black and white in the print edition. W Online version contains Web-only data. is regulated by many cues, including photoperiod. In Arabidopsis thaliana, long days (LDs) accelerate flowering, whereas short days (SDs) delay flowering (Liu et al., 2008). Genetic screening for Arabidopsis mutants involved in light-regulated seedling development followed by biochemical analyses identified a group of pleiotropic Constitutive Photomorphogenic/De-etiolated/Fusca (COP/DET/FUS) proteins that are central negative regulators of photomorphogenesis (Sullivan et al., 2003; Yi and Deng, 2005). These proteins are components of one of three biochemical entities: the COP1-SPA complexes, the COP9 signalosome (CSN), or the CDD complex (COP10, DDB1, and DET1), all of which are involved in proteasomal degradation of photomorphogenesis-promoting factors (Serino and Deng, 2003; Yanagawa et al., 2004; Yi and Deng, 2005; Zhu et al., 2008). COP1 is a conserved Ring finger E3 ubiquitin ligase that is involved in multiple processes in many different organisms, including plant development and mammalian cell survival, growth, and metabolism. COP1 was first cloned and characterized in the model plant Arabidopsis as a repressor of light-regulated plant development (Deng et al., 1991, 1992). COP1 was shown to act as an E3 ligase that targets several photomorphogenesispromoting proteins for destruction, including ELONGATED HYPOCOTYL5 (HY5; Osterlund et al., 2000), HY5 Homolog

2 CUL4-DDB1 Associates with COP1-SPA Complexes 109 (Holm et al., 2002), LONG AFTER FAR-RED LIGHT1 (Seo et al., 2003), LONG HYPOCOTYL IN FAR-RED1 (Duek et al., 2004; Jang et al., 2005; Yang et al., 2005), and Phytochrome A (Seo et al., 2004). Recently, COP1 has also been identified as an E3 ligase involved in the regulation of flowering time by directly targeting transcriptional activator CONSTANS (CO) for degradation (Jang et al., 2008; Liu et al., 2008). The expression of FLOWERING LOCUS T (FT) is highly and rapidly increased in response to CO expression (Samach et al., 2000). Also, COP1 can act with the substrate adaptor EARLY FLOWERING 3 (ELF3) to modulate light input to the circadian clock by destabilizing GIGANTEA (GI) (Yu et al., 2008). SUPPRESSOR OF PHYA 1 (SPA1) was first identified as a repressor of PHYA (Hoecker et al., 1998). In Arabidopsis, there are three additional SPA1-like proteins: SPA2, SPA3, and SPA4 (Hoecker et al., 1998, 1999; Zhu et al., 2008). The quadruple spa mutant displays a phenotype similar to that of strong cop1 alleles, and the SPA genes are partially redundant in mediating light responses at both seedling and adult stages (Laubinger and Hoecker, 2003; Laubinger et al., 2004, 2006). Biochemical analysis demonstrated that the SPA proteins can self-associate or interact with each other, forming a heterogeneous group of SPA- COP1 complexes in Arabidopsis (Zhu et al., 2008). CULLIN4 (CUL4), a recently identified regulator of photomorphogenesis, links the three COP complexes (the COP1-SPA complexes, CSN, and the CDD complex) in the ubiquitination/ proteasome-mediated degradation of key photomorphogenesis-promoting factors (Chen et al., 2006). CUL4 is an important member of the Cullin family and serves as a scaffold in CUL4- DDB1 based ubiquitin ligases to regulate many processes, including cell proliferation, DNA repair, and genomic integrity by ubiquitinating key regulators (Lee and Zhou, 2007). Recent studies have identified a group of DDB1 and CUL4-Associated Factors (DCAFs) or DDB1 binding WD40 proteins (DWD) as substrate receptors that dictate the specificity of the ubiquitination process in different organisms (Angers et al., 2006; He et al., 2006; Higa et al., 2006b; Jin et al., 2006; Lee et al., 2008; Zhang et al., 2008). Furthermore, it has been demonstrated that several of these DCAFs or DWD proteins interact with RING-BOX1 (RBX1)/Regulator of Cullins 1 (ROC1)-CUL4-DDB1 to form functional E3 ligases. For example, CDT2 was shown to interact with the CUL4-DDB1 complex to regulate S phase destruction of the replication factor CDT1 in response to DNA damage (Higa et al., 2006a; Jin et al., 2006; Ralph et al., 2006; Sansam et al., 2006). It was further demonstrated that the CUL4-DDB1-CDT2 ubiquitin ligase also targets the CDK inhibitor p21 for ubiquitination and degradation (Abbas et al., 2008; Kim et al., 2008; Nishitani et al., 2008). Similarly, DCAF1/VprBP, a WD40 protein, forms a nuclear E3 ubiquitin ligase with CUL4-DDB1 and regulates DNA replication and embryonic development in both mammalian and plant systems (McCall et al., 2008; Zhang et al., 2008). Related studies identified the CUL4-DDB1-VprBP E3 ubiquitin ligase complex as the downstream effector of lentiviral Vpr in arresting the cell cycle in G2 phase (Hrecka et al., 2007; Le Rouzic et al., 2007). Moreover, WD40 protein FBW5 (for F-box and WD repeat domain containing 5) was shown to promote ubiquitination of tumor suppressor TSC2 and regulate TSC2 protein stability by associating with DDB1-CUL4-ROC1 ligase (Hu et al., 2008). Similarly, in plants, DWD protein PRL1 was identified as the substrate receptor of a CUL4-ROC1-DDB1-PRL1 E3 ligase involved in the degradation of AKIN10 (Lee et al., 2008). Our previous work showed that CUL4 may interact with the CDD complex to form an E3 ligase and regulate photomorphogenesis. In addition, CUL4 associated with COP1, which suggested that these two proteins may work together to regulate photomorphogenesis (Chen et al., 2006). Recently, a detailed biochemical characterization of the COP1-SPA complexes suggested that the four endogenous homologous SPA proteins can form stable complexes with COP1 in vivo regardless of light conditions, although individual SPA proteins exhibited distinct expression profiles in different tissues and light conditions (Zhu et al., 2008). However, the mechanism by which CUL4-DDB1 works with the COP1-SPA complexes to mediate the light regulation of plant development remains to be elucidated. Here, we demonstrate that CUL4-DDB1 directly interacts with the COP1-SPA complexes in vivo and in vitro and that this interaction still exists in the absence of the CDD complex in Arabidopsis. CUL4-DDB1 and COP1-SPA complexes may work in concert to regulate photomorphogenesis and, possibly, flowering time under SD conditions. Furthermore, we hypothesize that CUL4-DDB1 may interact with COP1-SPA complexes to form a new group of E3 ligases, which are distinct from the previously described RBX1-CUL4-CDD complex but work in concert with this complex to mediate light regulation of plant development. RESULTS Both COP10 and DET1 Are Required for the Normal Accumulation of the CDD Complex Previous data showed that COP10 is significantly reduced and only present as monomers in det1-1 mutants (Pepper et al., 1994; Yanagawa et al., 2004). To gain further insight into the relationship between COP10 and DET1 proteins, we first measured the steady state abundance of DET1 in 6-d-old cop10-1 (Suzuki et al., 2002), det1-1, and wild-type Wassilewskija (Ws) and Columbia (Col) Arabidopsis seedlings grown in darkness and continuous white light. As shown in Figure 1A, DET1 abundance is dramatically reduced in cop10-1 and absent in det1-1 under both light and dark conditions. We next performed gel filtration analysis to investigate formation of the DET1-containing complex in 6-d-old light-grown wild-type, cop10-1, and 35S:flag- COP10 (in cop10-1 mutant background) seedlings. As shown in Figure 1B, DET1 was most abundant in the 200- to 300-kD fractions in wild-type Arabidopsis seedlings, consistent with the size of the CDD complex purified previously (Yanagawa et al., 2004). Interestingly, the quantity of the DET1-containing complex was reduced in the cop10-1 mutant (Figure 1B). The CDD complex was restored to normal levels in the 35S:flag-COP10 transgenic line, which completely complements the hyperphotomorphogenic phenotype caused by the absence of COP10 in cop10-1. Thus, our results indicate that COP10 is essential for normal CDD complex accumulation. In contrast with these results with DET1, previous data showed that the DDB1 gel filtration profile is very similar in the wild type

3 110 The Plant Cell Figure 1. CUL4 and COP1 Interact with Each Other in the Absence of the CDD Complex. (A) The DET1 protein level is dramatically reduced in cop10-1 mutants. Total protein extracts from 6-d-old continuous white light grown (cw) and darkgrown (cd) seedlings were examined by protein gel blot analysis using anti-cul4, anti-det1, and anti-tubulin. Tubulin protein level was used as a sample loading control. (B) Protein gel blot analyses showing the gel filtration patterns of DET1 and CUL4 proteins in Ws (wild type), cop10-1, and flag-cop10 (in cop10-1 background), respectively. Molecular masses are indicated at bottom. T, total unfractionated extracts. (C) Flag-CUL4 associates with DDB1 and COP1 in vivo in wild-type, cop10-1, and det1-1 backgrounds. Total protein extracts prepared from 6-d-old dark-grown wild-type, 35S:flag-CUL4, 35S:flag-CUL4/cop10-1, and 35S:flag-CUL4/det1-1 transgenic Arabidopsis seedlings were incubated with antiflag antibody-conjugated agarose (a-flag). The precipitates and total extracts were subjected to immunoblot analysis with antibodies against flag, DDB1, DET1, COP1, and RPN6. and cop10-1 mutants (Yanagawa et al., 2004). Similarly, we found the CUL4 gel filtration profiles were quite similar in Ws (wild-type), cop10-1, and flag-cop10, and the abundance of CUL4 in cop10-1 was almost unchanged as well (Figures 1A and 1B). Thus, CUL4-DDB1 is probably involved in other protein complexes in addition to the CUL4-DDB1-CDD complex. CUL4 and COP1 Interact with Each Other in the Absence of the CDD Complex The fact that RBX1-CUL4 can interact with the CDD complex to form an E3 ligase (Chen et al., 2006) and that COP10 and COP1 can associate in vivo (Yanagawa et al., 2004) led us to further study interactions between CUL4-DDB1 and COP1-containing complexes. To test the in vivo interaction between CUL4 and COP1, we performed coimmunoprecipitation analyses on 6-d-old dark-grown wild-type, 35S:flag-CUL4, 35S:flag-CUL4/ cop10-1, and 35S:flag-CUL4/det1-1 seedlings, respectively. In the det1-1 background, all COP10 was in monomer form, and the CDD complex was disrupted (Yanagawa et al., 2004). As shown in Figure 1C, CUL4 associates with DDB1 and COP1 in the absence of COP10 or DET1, showing that the existence of the CDD complex is not necessary for CUL4 to interact with COP1. DDB1 Interacts with COP1 and SPA Proteins in Vitro Recently, several reports showed that a group of proteins containing the WDXR motif (DWD or DCAF proteins) may work

4 CUL4-DDB1 Associates with COP1-SPA Complexes 111 as adaptors that interact with DDB1 in Cullin-based E3 ligases and recruit the substrates for degradation in both mammalian and plant systems (He et al., 2006; Higa et al., 2006b; Jin et al., 2006; Lee et al., 2008). Sequence alignment analysis showed that all known components of Arabidopsis COP1-SPA complexes contain at least one WDXR motif (Figure 2A; Lee et al., 2008). We therefore tested interactions of DDB1 with components of COP1-SPA complexes by in vitro pull-down assays. In these assays (Figure 2B), recombinant DDB1A and DDB1B glutathione S-transferase (GST) fusion proteins were used to pull down COP1 tagged with maltose binding protein (MBP) and 35 S Met-labeled his-tagged SPA proteins produced by coupled in vitro transcription/translation reactions. As shown in Figure 2B, GST-DDB1B could pull down MBP-COP1 and all His-tagged SPA proteins. The amounts of MBP-COP1 and His-SPA proteins pulled down by GST-DDB1A were lower, possibly due to the instability and limited amount of GST-DDB1A in our system. To further test whether DDB1 interacts with COP1 complex components via the WDXR motif, the following amino acids were mutated in the WDXR motifs of COP1, SPA1, and SPA3 (three representatives showing clear interactions with GST-DDB1B in Figure 2B): D534A, R536A, D576A, and K578A in COP1, D879A and R881A in SPA1, and D689A and R691A in SPA3. As expected, the mutations in COP1, SPA1, and SPA3 disrupted their interactions with GST-DDB1B (Figure 2C; see Supplemental Figure 1 online), which demonstrates that the WDXR motifs of COP1 and SPAs are critically important for interaction with DDB1. In Vivo Association between CUL4-DDB1 and COP1-SPA Complexes Arabidopsis has two isoforms of DDB1: DDB1A and DDB1B. DDB1B is the predominant player in regulating embryogenesis since knockout line ddb1b is embryo-lethal, while knockout line ddb1a exhibits no obvious phenotype (Schroeder et al., 2002). DDB1 works with CUL4 by directly interacting with both CUL4 and downstream adaptor proteins, and CUL4 and COP1 have been observed to associate in vivo in Arabidopsis (Angers et al., 2006; Chen et al., 2006; He et al., 2006; Higa et al., 2006b; Jin et al., 2006; Lee et al., 2008). We therefore tested whether DDB1B interacts with COP1-SPA complexes by performing coimmunoprecipitation analyses using transgenic ddb1a seedlings transformed with full-length DDB1B fused to the flag peptide and driven by the 35S promoter (35S:flag-DDB1B/ ddb1a) grown for 6 d in the dark or in continuous white light. Transgenic flag-ddb1b pulled down CUL4, DET1, COP1, and SPA1 (Figure 3A), which suggested that CUL4-DDB1 and COP1- SPA complexes associate in vivo. Since the Arabidopsis SPA family has four members, which have all been shown to interact with COP1 in all light conditions and form heterogeneous complexes (Zhu et al., 2008), we further tested the interactions of DDB1 with SPA proteins using seedlings transformed with SPA proteins fused to the tandem affinity purification (TAP) peptide in the appropriate mutant background. All four TAP-SPA proteins pulled down COP1 very effectively, confirming that COP1 and SPA proteins may form tight complexes (see Supplemental Figure 2 online). TAP-SPA1, TAP-SPA3, and TAP-SPA4 also coimmunoprecipitated low levels of DDB1 protein (see Figure 2. DDB1A and DDB1B Interact with COP1 and SPA Proteins in Vitro. (A) Alignment of the WDXR regions of COP1, SPA1, SPA2, SPA3, and SPA4. The WDXR motif is highlighted in red. Pink, conserved D or E; yellow, hydrophobic amino acids; gray, small amino acids. (B) In vitro coimmunoprecipitation of COP1 and SPA proteins by recombinant DDB1A and DDB1B. Recombinant MBP-COP1 purified from Escherichia coli and 35 S-labeled His-SPA1, His-SPA2, His-SPA3, and His-SPA4 generated by the TNT system were used as prey molecules and incubated with unlabeled GST, GST-DDB1A, and GST-DDB1B, respectively. Anti-GST antibody fused to agarose was used for immunoprecipitation. Pellet fractions were resolved by SDS-PAGE and visualized by Coomassie blue staining (for GST fused proteins), anti-cop1 immunoblot (for MBP-COP1), and autoradiography using a phosphor imager (for SPA proteins). Quantification was based on normalization against the value of the background region. Band intensities of MBP- COP1, His-SPA1, His-SPA2, His-SPA3, and His-SPA4 pulled down by GST-DDB1B were each set to 100. Relative band intensities were then calculated and are indicated by numbers below blots. (C) Point mutations within the WDXR motifs of COP1 and SPA proteins disrupt their interactions with DDB1. MBP-COP1mWDXR contains the following mutations in the WDXR motifs: D534A, R536A, D576A, and K578A; His-SPA1mWDXR contains mutations D879A and R881A; His- SPA3mWDXR contains mutations D689A and R691A. Wild-type and mutated recombinant MBP-COP1 purified from E. coli, and wild-type and mutated 35 S-labeled His-SPA1, His-SPA3 generated by the TNT system were used as prey molecules and incubated with GST and GST- DDB1B, respectively. Anti-GST antibody fused to agarose was used for immunoprecipitation. Pellet fractions were resolved by SDS-PAGE and visualized by anti-gst immunoblot (for GST fused proteins), anti-mbp immunoblot (for wild-type and mutated MBP-COP1), and autoradiography using x-ray films (for wild-type and mutated SPA proteins). Quantification was performed for each pair of wild-type and mutated prey proteins. Band intensities of MBP-COP1, His-SPA1, and His-SPA3 pulled down by GST-DDB1B were set to 100. Relative band intensities were then calculated and are indicated by numbers below blots.

5 112 The Plant Cell Figure 3. CUL4-DDB1 Interacts with the COP1-SPA Complexes in Vivo. (A) Flag-DDB1B associates with CUL4, DET1, COP1, and SPA1 in vivo. Total protein extracts prepared from 6-d-old continuous white light grown (cw) and dark-grown (cd) wild-type and 35S:flag-DDB1B/ddb1a (abbreviated as flag-ddb1b) transgenic Arabidopsis seedlings were incubated with antiflag antibody-conjugated agarose (a-flag). The precipitates and total extracts were subjected to immunoblot analysis with antibodies against flag, CUL4, DET1, COP1, SPA1, and RPN6. RPN6 was used as a control. (B) Flag-DDB1B associates with COP1 in wild-type and det1-1 backgrounds. Total protein extracts prepared from 6-d-old dark-grown wild-type, 35S: flag-ddb1b/ddb1a (abbreviated to be flag-ddb1b), and 35S:flag-DDB1B/det1-1 transgenic Arabidopsis seedlings were incubated with anti-flag antibody-conjugated agarose (a-flag). The precipitates and total extracts were subjected to immunoblot analysis with antibodies against flag, DET1, COP1, and RPN6. RPN6 was used as a control. Supplemental Figure 2 online). The low level of DDB1 pulled down by SPA proteins is probably due to the fact that DDB1 can interact with a large number of different downstream adaptor proteins (Lee et al., 2008; Zhang et al., 2008). TAP-SPA2 failed to pull down DDB1, probably due to the low abundance of TAP- SPA2 and the presence of truncated SPA2 in that line, which likely competes with TAP-SPA2 for interaction with DDB1. Although the interactions shown between TAP-SPA1, TAP-SPA3, TAP-SPA4, and DDB1 are weak (see Supplemental Figure 2 online), this result does support the interactions between DDB1 and SPA proteins shown in Figures 2 and 3A. To further test the interaction between CUL4-DDB1 and COP1 complexes, flag- DDB1B transformed into the det1-1 mutant was used for coimmunoprecipitation. As shown in Figure 3B, flag-ddb1b can still pull down COP1 in the absence of DET1, similarly to flag-cul4 (Figure 1C), further demonstrating that the interaction between CUL4-DDB1 and COP1 exists in the absence of the CDD complex. CUL4-DDB1 and COP1-SPA Complexes Act Together to Regulate Photomorphogenesis Since both in vivo and in vitro data showed that there are biochemical interactions between CUL4-DDB1 and the COP1-SPA complexes, we decided to investigate the functional significance of this biochemical interaction in plant development. To this end, we crossed a weak cop1 allele, cop1-4 (McNellis et al., 1994), with cul4cs (for CUL4 cosuppression; Chen et al., 2006) and examined the photomorphogenic phenotype. Some cul4cs plants (<5%) opened their cotyledons and had short hypocotyls in the dark, which are characteristics of weak constitutive photomorphogenic mutants. We found that 6-d-old dark-grown cul4cs cop1-4 seedlings had opened cotyledons and extremely short hypocotyls, which is similar to the cop lethal phenotype (Figure 4A). Moreover, these double mutants accumulated large amounts of anthocyanin, similar to the null mutant cop1-5 (McNellis et al., 1994). In addition, 25% of the F2 seeds of cul4cs crossed with cop1-4 were purple (Figure 4B) due to the anthocyanin overaccumulation typically found in cop lethal mutants (McNellis et al., 1994). Thus, reduction of CUL4 function enhances the weak cop1-4 mutant phenotype, supporting the hypothesis that CUL4-DDB1 works together with COP1-SPA complexes to regulate plant photomorphogenesis. CUL4 Regulates Flowering Time under SD Conditions Previous data showed that both COP1 and SPA1 are involved in regulating flowering time, and both cop1 and spa1 mutants

6 CUL4-DDB1 Associates with COP1-SPA Complexes 113 Figure 4. CUL4 and COP1 Function Together to Regulate Photomorphogenesis. (A) Repression of CUL4 enhances the seedling phenotype of the weak cop1-4 allele in the dark. Different Arabidopsis lines (labeled at bottom) were grown in complete darkness for 6 d. Bars = 1 mm. (B) Repression of CUL4 increases anthocyanin accumulation in cop1-4 seeds. The cul4cs and cop1-4 seeds are homozygous, while the cul4cs cop1-4 are F2 seeds from hybrid F1 plants. flower earlier under SDs (8 h light/16 h dark) (McNellis et al., 1994; Laubinger et al., 2006; Jang et al., 2008; Liu et al., 2008). Since CUL4 associates with COP1 and SPA1, we next asked whether CUL4 is involved in regulating flowering time under SDs and found that cul4cs flowered much earlier than its counterpart wildtype Col under SDs (Figure 5A, Table 1). When the siliques of cul4cs turned yellow, the wild-type plants had not yet bolted. This result indicated that CUL4 is functionally involved in regulating flowering time. By contrast, under LDs (16 h light/8 h dark) cul4cs plants flowered at the same time as wild-type Col (Table 1). Thus, CUL4 regulates flowering time specifically under SDs. In previous studies on photoperiodic flowering, CO was identified as a pivotal regulator for triggering flowering, which induces the expression of the flowering time gene FT (Putterill et al., 1995; An et al., 2004). Early flowering of SD-grown spa mutants correlates with strongly increased FT transcript levels, whereas CO transcript levels are not altered, and SPA proteins regulate the stability of CO protein (Laubinger et al., 2006). COP1 regulates CO function at both transcriptional and posttranslational levels and reduces FT mrna levels (Jang et al., 2008; Liu et al., 2008; Yu et al., 2008). To investigate whether CUL4 also influences CO and FT, CO and FT transcript levels were examined in Col and cul4cs grown under SDs and harvested at Zeitgeber (ZT) 16 (Figure 5B). There was no obvious difference in CO mrna abundance between cul4cs and its wild-type counterpart Col. By contrast, FT transcript levels in 20-d-old cul4cs were higher than in Col. We therefore studied the time course of FT transcription, starting with young seedlings under SDs, by quantitative realtime PCR. As shown in Figure 5C, compared with in Col, FT transcription in cul4cs was elevated in the fourth day and dramatically increased from the sixth day after they were sown in soil. Thus, the decreased level of CUL4 protein resulted in a significant increase in FT transcript level starting early in development, indicating that CUL4 may play a negative role in regulating FT transcription. Since transcription of CO and FT shows circadian regulation, we examined CO and FT transcript levels in wild-type and cul4cs plants to test whether CUL4 affects the circadian patterns of CO and FT expression. As shown in Figure 5D, wild-type and cul4cs plants showed very similar diurnal regulation of CO mrna levels in SD-grown plants. Interestingly, FT transcript levels in cul4cs were elevated through the whole day with two possible minor peaks (Figure 5E). The pattern with a peak level of FT transcripts in cul4cs around ZT 20 is similar to the patterns in cop1 and spa mutants (Laubinger et al., 2006; Yu et al., 2008), which indicates that CUL4 may function similarly to the COP1 and SPA proteins to regulate FT transcription during the night phase. However, the phenomenon that FT transcript levels increased during the day phase with a possible minor peak level around ZT 8 is new. The possible two-peak pattern of FT transcripts in cul4cs was consistent in the two continuous days that we examined and was repeatable in three independent experiments. The mechanism of the regulation of FT transcripts by CUL4 is unclear. DDB1 Is A Core Adaptor Protein In CUL4-based E3 ligases, DDB1 plays an important role in recruiting the adaptor proteins. DDB1 has three domains and interacts with CUL4 via the BPB domain (Angers et al., 2006; Jin et al., 2006). To test specific roles of the DDB1 domains in Arabidopsis, we generated three groups of point mutations on the surface of DDB1B s three domains, which are predicted to be responsible for mediating interactions with other proteins based on structural information. The mutated amino acids and their positions on the surface of DDB1B are shown in Figure 6A. Mutant DDB1B coding sequences tagged with the FLAG peptide and driven by the 35S promoter were transformed into ddb1a plants, and the three resulting groups of transgenic plants were named mbpa, mbpb, and mbpc, corresponding to the mutations within the BPA, BPB, and BPC domains of DDB1B. Transgenic mbpa and mbpb plants had phenotypes similar to ddb1a plants and were used for coimmunoprecipitation analyses. As shown in Figure 6B, the mutations within the BPB domain completely disrupted the interaction between CUL4 and DDB1, while the mutations within the BPA domain did not affect the interaction between CUL4 and DDB1. This is consistent with results in mammalian cells (Angers et al., 2006; Jin et al., 2006). Mutating the BPC domain had a very different effect: several mbpc transgenic lines showed a severely dwarfed phenotype (Figure 6C) that was stably transmitted to the next generation, suggesting that the BPC domain is important for DDB1 function. Although we are interested in studying the effects of the BPC domain mutation on DDB1 s interactions with other proteins, we could not collect enough material for further coimmunoprecipitation analysis because each dwarf mbpc plant only yields a few seeds. The observation that 75% of the progeny from a heterozygous mbpc plant showed the identical severely dwarfed phenotype indicates that one copy of the transgenic fragment is

7 114 The Plant Cell sufficient to induce the phenotype. To test whether the dwarf phenotype of mbpc is due to cosuppression of DDB1B, we tested the levels of DDB1B mrna and protein and found that both DDB1B mrna and protein levels, including endogenous DDB1B and transgenic mutated DDB1B, are higher in mbpc than in ddb1a (Figures 6D and 6E). Accordingly, the dwarf phenotype may result from a dominant-negative effect of the mutated DDB1B protein rather than DDB1B cosuppression. No Obvious in Vivo Association between the CDD and COP1-SPA Complexes Figure 5. CUL4 Is Involved in the Regulation of Flowering Time under SDs. (A) cul4cs flowers early in SDs. cul4cs and its wild-type counterpart Col were grown for 90 d under SDs (8 h light/16 h dark). (B) Levels of CO and FT transcripts in cul4cs and wild-type Col. Total RNA samples were collected from 20-d-old Col and cul4cs plants grown under SDs (8 h light/16 h dark) and harvested at ZT 16. mrna levels of CO, FT, and ACTIN2 were tested by RT-PCR with three repeats. ACTIN2 transcripts were used as control. (C) FT transcription starts very early in SD-grown cul4cs. Total RNA samples were collected from 4-, 6-, 8-, 10-, 12-, and 14-d-old Col and cul4cs plants grown under SDs (8 h light/16 h dark) and harvested at ZT 16. Levels of FT mrna and 18S rrna were tested by real-time PCR. Although COP1-SPA complexes can interact with CUL4-DDB1 in the absence of the CDD complex, it is still possible that CUL4- DDB1, COP1-SPA complexes, and the CDD complex all exist in a super complex connected by DDB1 s multiple interacting domains in planta. To test this, the interaction between DET1 and COP1, core components of the CDD complex and COP1- SPA complexes, respectively, was examined by coimmunoprecipitation. Because native DET1 protein runs close to the IgG heavy chain in SDS-PAGE and it is difficult to distinguish from the IgG heavy chain, transgenic green fluorescent protein (GFP)- DET1/det1-1 that rescued the det1-1 phenotype was used for testing the interaction between DET1 and COP1. As shown in Figure 7A, using seedlings grown under both white light and dark conditions, antibodies against DET1 precipitated GFP-DET1 very well but did not pull down COP1 at all. Conversely, COP1 antibodies precipitated COP1 very well but did not pull down GFP-DET1. At the same time, both DET1 and COP1 showed interactions with DDB1. In addition, we used TAP-COP1 seedlings (transformed with COP1 fused to the TAP peptide) to confirm the result that COP1 and DET1 have no obvious interaction. As shown in Figure 7B, TAP-COP1 could pull down DDB1 well but failed to pull down DET1, confirming that COP1 is associated with DDB1 but not DET1 in planta. Thus, we did not detect an in vivo association between DET1 and COP1 proteins, indicating that CDD and COP1-SPA complexes may not exist in a supercomplex. We next performed gel filtration analysis to investigate formation of the DET1-containing complex in 6-d-old light-grown wildtype and quadruple spa (Laubinger et al., 2006) seedlings. As shown in Figure 7C, DET1 was found in similar fractions in extracts from wild-type and spa1234 mutants, which suggests that integrity of DET1-containing complexes is not dependent on COP1-SPA complexes. Similarly, formation of COP1-containing complexes in 6-d-old light-grown wild-type and det1-1 seedlings was tested by gel filtration. As shown in Figure 7C, COP1 Relative amounts of FT transcripts were normalized to the levels of 18S rrna in the same sample. (D) and (E) Circadian patterns of CO (D) and FT (E) mrna levels in cul4cs and wild-type Col. The plants were grown under SDs (8 h light [white bar]/ 16 h dark [black bar]) for 18 d and then RNA samples were collected every 4 h, at the times shown, after dawn. Levels of CO and FT mrna and 18S rrna were analyzed by real-time PCR. Relative amounts of CO and FT transcripts were normalized to the levels of 18S rrna. For (C) to (E), mean and SD values of four replicates are shown. Some error bars are too small to be shown in the figure.

8 CUL4-DDB1 Associates with COP1-SPA Complexes 115 Table 1. Flowering Time of cul4cs and Col in SDs and LDs Genotype Number of Leaves (SD) Number of Leaves (LD) Col (wild type) cul4cs Plants were grown in SDs (8 h light/16 h dark) and LDs (16 h light/8 h dark). Values shown are mean numbers of rosette leaves at flowering 6 SD. At least 15 plants were analyzed for each genotype. The cul4cs mutation is in the Col ecotype. complexes in the absence of DET1 are similar to those in the wild type, which indicates that the COP1-containing complexes exist in the absence of the DET1-containing complexes. These findings support the hypothesis that CDD and COP1-SPA are distinct complexes. Given that flag-cop10 can coimmunoprecipitate COP1 in vivo (Yanagawa et al., 2004), we then examined whether the in vivo association between flag-cop10 and COP1 supports an interaction between the CDD complex and COP1-SPA complexes. To do so, we performed gel filtration and coimmunoprecipitation on 35S:flag-COP10/det1-1 extracts. The gel filtration profile of flag-cop10 in det1-1 indicated that it existed solely as monomers in this background, whereas it existed as both monomers and in complexes in cop10-1 (Figure 7D). This is consistent with the result that endogenous COP10 exists solely as monomers in det1-1 mutants (Yanagawa et al., 2004). Coimmunoprecipitation showed that flag-cop10 still associates with COP1 when DET1 is disrupted (Figure 7E). Therefore, it is the COP10 monomer rather than the CDD complex that associates with COP1. Accordingly, interaction between COP10 and COP1 does not indicate that the CDD complex and the COP1-SPA complexes interact. Instead, these data support a conclusion that both the CDD complex and the COP1-SPA complexes can associate with CUL4 in vivo but may form distinct complexes instead of a supercomplex. CDD and COP1-SPA Complexes Act Additively to Regulate Photomorphogenesis To characterize the functional relations between the CDD complex and COP1-SPA complexes, the weak alleles cop10-4 and det1-1 were introduced into the spa triple mutants. As shown in Figure 8, reduction of COP10 and DET1 additively enhances the phenotype of spa triple mutants, which is consistent with the lethal phenotype of the double mutant det1-1 cop1-6 (Ang and Deng, 1994). This shows that the components of the CDD complex and the COP1-SPA complexes have genetic interactions, implying that even without a direct association they may work closely together in regulating photomorphogenesis. DISCUSSION This study provides a biochemical and functional analysis of CUL4-DDB1 and COP1-SPA complexes. Our data show that CUL4-DDB1 may directly interact with COP1-SPA complexes in the absence of the CDD complex and form a heterogenous group of E3 ligases that regulate multiple aspects of the light regulation of plant development. Our previous data showed that the CSN regulates the rubylation and derubylation cycle of CUL4, while the CDD complex may bind CUL4 to form another E3 ligase (Chen et al., 2006). How this CUL4-CDD E3 ligase works together with CUL4-DDB1-COP1-SPA E3 ligases remains to be elucidated. CUL4-DDB1 and COP1-SPA Complexes Associate in Vivo Previous data showed that CUL4 and COP1 proteins interact, indicating that they may work in concert to mediate repression of photomorphogenesis (Chen et al., 2006); here, we provide more evidence to reveal the mechanism by which CUL4-DDB1 and COP1-SPA complexes work together. Recently, several groups independently reported that CUL4-based E3 ligases use the adaptor protein DDB1 to interact with DCAF substrate recognition receptors (also called DWD or CDW proteins) and recruit the substrates (Angers et al., 2006; He et al., 2006; Higa et al., 2006b; Jin et al., 2006; Lee et al., 2008). Interestingly, core components of COP1-SPA complexes, including COP1, SPA1, SPA2, SPA3, and SPA4, all contain the WDXR motif, which was suggested to be the determinant feature for binding DDB1. In vitro pull-down experiments demonstrated that COP1 and SPA proteins may directly interact with DDB1 (Figure 2), while in vivo coimmunoprecipitation showed that the components of these COP1-SPA complexes are associated with CUL4-DDB1 (Figure 3). Point mutations within the WDXR motifs of MBP-COP1, His-SPA1, and His-SPA3 disrupted their interactions with GST-DDB1B, which suggests that associations between CUL4-DDB1 and COP1- SPA complexes are via WDXR motifs. Regulation of these interactions by different light conditions needs further study. CUL4-DDB1 and COP1-SPA Complexes Work Together to Regulate Multiple Aspects of the Light Regulation of Plant Development Since cul4cs, cop1, and spa mutants all show similar photomorphogenetic phenotypes (Deng et al., 1991, 1992; Laubinger et al., 2004; Chen et al., 2006), it is expected that they may work together to regulate the repression of photomorphogenesis. Genetic interaction tests showed that reduction of CUL4 resulted in enhancement of the phenotype of the weak cop1-4 mutant, and the phenotype of cul4cs cop1-4 double mutants was identical to the lethal mutant cop1-5 (Figure 4). This is consistent with previous data showing that CUL4 positively regulates the degradation of HY5, a transcription factor enhancing photomorphogenesis regulated by COP1-SPA1 (Osterlund et al., 2000; Saijo et al., 2003; Chen et al., 2006). We therefore conclude that CUL4- DDB1 and the COP1-SPA complexes may work together to regulate photomorphogenesis by regulating transcription factors such as HY5. As previously reported, cop1-4, spa1-7, and spa1 spa3 spa4 triple mutants exhibit early flowering phenotypes, and COP1 and SPAs are involved in regulating flowering time under SDs (McNellis et al., 1994; Laubinger et al., 2006; Jang et al., 2008; Liu et al., 2008). Since CUL4 interacts with COP1 and SPA1, we tested and found that cul4cs also flowers earlier than the wild

9 116 The Plant Cell Figure 6. Effects of Point Mutations on the Surface of the DDB1 Protein. (A) Structural diagram depicting the locations of the three groups of mutations on the surface of DDB1. Mutated residues are indicated. (B) Effects of DDB1 mutations on binding to CUL4 and COP1. Wild-type or point-mutated DDB1B coding sequences fused to the flag peptide and driven by the 35S promoter were transformed into ddb1a mutants, and the transgenic plants were used for coimmunoprecipitation. DDB1B, mbpa, and mbpb indicate wild-type flag-ddb1b, flag-ddb1b with point mutations within the BPA domain, and flag-ddb1b with point mutations within the BPB domain, respectively. Total protein extracts from 6-d-old light-grown Arabidopsis seedlings were incubated with anti-flag antibody-conjugated agarose (a-flag). The precipitates and total extracts were subjected to immunoblot analysis with antibodies against flag, CUL4, and COP1. (C) Phenotypes of 38-d-old ddb1a and mbpc plants under LDs (16 h light/8 h dark). The ddb1a mutant is in the left pot, and the mbpc mutant is in the right pot. mbpc contains flag-ddb1b with point mutations in the BPC domain transformed into the ddb1a mutant background. (D) DDB1 transcript levels in ddb1a and mbpc. Total RNA samples were collected from 4-week-old ddb1a and mbpc plants, and DDB1B expression levels were quantified by RT-PCR with three repeats. ACTIN2 transcripts were used as control. (E) DDB1 protein levels in ddb1a and mbpc. Total proteins were extracted from 15-d-old ddb1a and mbpc plants and subjected to immunoblot analysis with antibodies against flag, DDB1, and Tubulin. Tubulin was used as a sample loading control.

10 CUL4-DDB1 Associates with COP1-SPA Complexes 117 Figure 7. Biochemical Interactions between the CDD and COP1-SPA Complexes. (A) GFP-DET1 and COP1 have no obvious in vivo interaction. Total protein extracts prepared from 6-d-old GFP-DET1/det1-1 transgenic Arabidopsis seedlings grown in continuous white light (cw) or in the dark (cd) were incubated with anti-cop1 or anti-det1 and then precipitated with Protein A agarose. The precipitates and total extracts were subjected to immunoblot analysis with antibodies against COP1, DET1, DDB1. and RPN6. (B) TAP-COP1 and DET1 have no obvious in vivo interaction. Total protein extracts prepared from 6-d-old Col and TAP-COP1 Arabidopsis seedlings grown in the dark or continuous white light were incubated with IgG-coupled Sepharose. The precipitates and total extracts were subjected to immunoblot analysis with antibodies against Myc, DDB1, DET1, and RPN6. RPN6 was used as a control. (C) Protein gel blot analyses showing the gel filtration patterns of DET1 protein in Col (wild type) and spa1234, and COP1 protein in Col (wild type) and det1-1. Molecular masses are indicated below the blot. Total: total unfractionated extracts.

11 118 The Plant Cell type (Figure 5A). Molecularly, the FT transcript level is elevated in cop1-4, spa1-7, and spa1 spa3 spa4 triple mutants (Laubinger et al., 2006; Jang et al., 2008; Liu et al., 2008). Here, cul4cs early flowering is ascribed to the accumulation of FT transcripts (Figures 5B and 5C), suggesting that CUL4 participates in the transcriptional regulation of FT and is involved in the negative regulation of flowering. As a floral output gene, FT mrna abundance is dependent on the level of CO protein (Valverde et al., 2004). It has been reported that both COP1 and SPA1 interact with CO and shape its temporal expression pattern by regulating its stability (Laubinger et al., 2006; Jang et al., 2008; Liu et al., 2008). CO is a positive regulator of flowering. The level of CO protein is elevated in spa1 mutants, whereas CO mrna levels are unchanged, suggesting that SPA proteins mediate CO protein degradation (Laubinger et al., 2006). Two recent reports demonstrated that COP1 can interact with CO directly and mediate its ubiquitination and degradation to regulate flowering time (Jang et al., 2008; Liu et al., 2008). Also, COP1 was shown to act with ELF3 to regulate GI stability and modulate expression of CO (Yu et al., 2008). Taken together, these data indicate that COP1-SPA complexes modulate flowering time by regulating CO function at both transcriptional and posttranscriptional levels (Yu et al., 2008). It is of interest to examine CUL4 s role in the regulation of CO. As shown in Figure 5D, CUL4 has no effect on CO transcript levels under SDs, which is similar to the situation that CO transcript levels are not altered in SD-grown spa mutants (Laubinger et al., 2006). By contrast, the onset of CO expression in cop1-4 mutants shifted 4 h earlier, leading to elevated CO expression during daytime (Yu et al., 2008). This indicates that CUL4-DDB1 may work with COP1-SPA complexes to regulate the degradation of CO protein, and COP1 itself can interact with ELF3 to modulate CO transcription through targeted destabilization of GI. Further examination of CO protein levels in cul4cs may provide more information about how CUL4 regulates flowering time under SDs. A critically interesting result is the circadian pattern of FT transcript levels in cul4cs, which shows general elevation with possibly two minor diurnal peaks. The peak of FT transcript levels around ZT 20 in SD-grown cul4cs is very similar to the peaks in cop1 and spa mutants (Figure 5E; Laubinger et al., 2006; Yu et al., 2008). In this report, we demonstrated that CUL4-DDB1 associates with COP1 and SPA proteins (Figures 1 to 3). It is possible that CUL4-DDB1 interacts with COP1-SPA complexes to degrade CO protein and reduce FT transcripts in the night phase of SDs. There is a possible second peak of FT transcript levels around ZT 8 in SD-grown cul4cs (Figure 5E). As shown in Figure 5D, CO mrna levels are very low around ZT 8, which means CO translation must also be low at this time. Also, the photoreceptor Figure 8. The CDD Complex and COP1-SPA Complexes Together Mediate Plant Photomorphogenesis. (A) cop10-4 enhances the seedling phenotype of spa1 spa2 spa4 triple mutants in the dark. (B) Reduction of DET1 in the spa1 spa2 spa3 triple mutant enhances the seedling phenotype of spa1 spa2 spa3 in the dark. All plants (labeled at bottom) were grown in continuous darkness for 6 d. Bars = 1 mm. [See online article for color version of this figure.] phyb promotes the degradation of CO protein in the early day, which means that CO protein accumulated in the night and early morning will not persist until ZT 8 (Valverde et al., 2004; Jang et al., 2008). Therefore, the increase in FT transcript levels around ZT 8 in SD-grown cul4cs is not regulated by CO protein. This conclusion is consistent with the reports that FT acts partially downstream of CO and may also be mediated by other regulators (Kardailsky et al., 1999; Kobayashi et al., 1999). The mechanism of CUL4 s repression of FT transcription in the day phase in SD remains to be elucidated. CUL4-DDB1-COP1-SPA Complexes and the CUL4-CDD Complex Are Two Distinct Groups of Complexes Involved in Light Regulation of Plant Development Our previous data showed that in Arabidopsis RBX1, CUL4 and CDD form a complex that regulates photomorphogenesis (Chen et al., 2006). Our data indicate that CUL4-DDB1 can interact with COP1-SPA complexes in the absence of the CDD complex Figure 7. (continued). (D) Protein gel blot analyses showing the gel filtration patterns of flag-cop10 (F-COP10) in cop10-1 and det1-1. Molecular weights are indicated below the blot. T, total unfractionated extracts. Arrow indicates the flag-cop10 complexes. (E) Flag-COP10 monomer interacts with COP1 in vivo. Total protein extracts prepared from 6-d-old wild-type, flag-cop10/cop10-1, and flag-cop10/ det1-1 transgenic Arabidopsis seedlings grown in the dark were incubated with anti-flag antibody-conjugated agarose (a-flag). The precipitates and total extracts were subjected to immunoblot analysis with antibodies against flag, COP1, DET1, and RPN6.

12 CUL4-DDB1 Associates with COP1-SPA Complexes 119 (Figures 1 to 3). In addition, the coimmunoprecipitation results using GFP-DET1/det1-1 and TAP-COP1 plants showed that there is no obvious association between DET1 and COP1 proteins, which excludes the possibility that CUL4, CDD, and COP1- SPA all exist in a super complex (Figures 7A and 7B). Similar DET1-containing complexes in quadruple spa mutants and wildtype plants and similar COP1-containing complexes in det1-1 and wild-type plants further support this conclusion (Figure 7C). Both DET1 and COP1 can interact with DDB1 (Figures 3, 7A, and 7B), but they may do so in distinct complexes. Since flag-cop10 can coimmunoprecipitate COP1 in Arabidopsis, the CDD complex was considered to associate with COP1 complexes in vivo (Yanagawa et al., 2004). However, our data show that there is no obvious association between DET1 and COP1, which seems to contradict the previous conclusion. Repeated experiments showed that there is interaction between flag-cop10 and COP1 and that this interaction persists when DET1 is disrupted (Figures 7D and 7E). This indicates that the existence of the CDD complex is not necessary for interaction between flag-cop10 and COP1 and that interaction between flag-cop10 and COP1 does not represent interaction between the CDD complex and COP1 complexes. In mammalian systems, DET1 and COP1 were shown to form a dimer and work as CUL4-based E3 ligase adaptor proteins for some substrates including c-jun (Wertz et al., 2004). However, in plant systems, much data suggest that DET1 and COP1 work in distinct complexes based on their different subcellular locations (Pepper et al., 1994; von Arnim and Deng, 1994; Ang et al., 1998; Schroeder et al., 2002), protein complex sizes (Saijo et al., 2003; Yanagawa et al., 2004), and reconstitution results (Chen et al., 2006). The work reported here further showed that there is no obvious in vivo association between DET1 and COP1. These data support the conclusion that DET1 and COP1 exist in distinct complexes. Although the CDD and COP1-SPA complexes have no direct interaction, they work together to regulate photomorphogenesis (Figure 8). We propose that in Arabidopsis, CUL4-DDB1 interacts with COP1-SPA complexes to form a group of ligases distinct from the previously identified CUL4-CDD complex (Figure 9). These two groups of ligases may work in concert to modulate the light regulation of plant development by targeting photomorphogenesis- and possibly flowering-promoting factors, such as HY5 and CO, for degradation. The function of all of these ligases depends on the derubylation activity of the CSN. However, the relationship between CUL4-CDD and CUL4-DDB1-COP1-SPA complexes and the mechanism by which the interaction between the complexes is regulated still need further investigation. This may require a combination of different strategies and studies under different light conditions and development stages. Another challenge is to determine the cooperation between the RBX1-CUL4-DDB1 and COP1-SPA E3 ligase complexes. In mammalian systems, c-jun was thought to be ubiquitinated by a CUL4-based E3 ligase using COP1 as an adaptor, but the RING finger domain activity of COP1 was found not to be necessary for the ubiquitination (Wertz et al., 2004). For targeting and ubiquitinating p53, COP1 itself can work as an E3 ligase, and its RING activity is necessary for the ubiquitination (Dornan et al., 2004). This indicates that COP1 may function as an E3 ligase for some Figure 9. A Working Model of How CUL4 and the Three COP/DET/FUS Complexes Mediate Light Regulation of Plant Development. RBX1-CUL4 and the CDD complex form a functional E3 ligase, while RBX1-CUL4-DDB1 may interact with COP1-SPA complexes to form another group of E3 ligases. Rubylation and derubylation of these CUL4- based E3 ligases are regulated by the CSN complex, and these ligases regulate the degradation of downstream factors to mediate light regulation of plant development. The RBX1-CUL4-CDD complex and RBX1- CUL4-DDB1-COP1-SPA complexes may have regulatory relationships (shown by broken arrow), which require further study. DDA1 (for DET1, DDB1 associated 1) is shown as a small-sized component of the CDD complex. SPAx and SPAy represent homogenous or heterogeneous SPA proteins. A, B, and C represent the BPA, BPB, and BPC domains of DDB1, respectively. Ub represents Ubiquitin. E1 represents a ubiquitinactivating enzyme, and E2 represents a ubiquitin-conjugating enzyme. substrates and work as an adaptor protein of CUL4-DDB1 E3 ligases for other substrates. In Arabidopsis, complementation experiments showed that COP1 ( ) and COP1 ( ), two truncated COP1 proteins lacking the RING domain, could weakly rescue the cop1-5 lethal allele (Stacey et al., 2000). This suggests that the RING activity of COP1 may not be essential for targeting some substrates. This will be tested by introducing COP1 (C52S, C55S, C86S, and C89A) with point mutations in the RING motif shown to abolish the ligase activity (Seo et al., 2003, 2004) into the cop1-5 lethal allele to see whether it can partially rescue the phenotype. In addition, interaction of COP1 (C52S, C55S, C86S, and C89A) with CUL4-DDB1 and the COP1

13 120 The Plant Cell substrate degradation pattern will be examined. These investigations, together with other genetic, biochemical, and cell biological studies, will reveal how the RBX1-CUL4-DDB1 and COP1-SPA complexes cooperate. METHODS Plant Material and Growth Conditions Wild-type Arabidopsis thaliana plants used in this study were Col-0, Ws, and RLD ecotypes. Mutants and transgenic lines previously described are as follows: cul4cs (Chen et al., 2006), cop10-1 (Wei et al., 1994), cop10-4 (Suzuki et al., 2002), det1-1 (Chory et al., 1989), ddb1a (Schroeder et al., 2002), cop1-4 (McNellis et al., 1994), spa1-3 (Hoecker et al., 1998), spa1 spa2 spa3 and spa1 spa2 spa4 (Laubinger et al., 2004), GFP-DET1/det1-1 (Schroeder et al., 2002), 35S:flag-DDB1B/ddb1a (Lee et al., 2008), 35S:TAP-SPA1 (Saijo et al., 2003), 35S:TAP-SPA2, 35S: TAP-SPA3, 35S:TAP-SPA4 (Zhu et al., 2008), 35S:TAP-COP1 (Rubio et al., 2005), 35S:flag-CUL4 (Chen et al., 2006), and 35S:flag-COP10 (Yanagawa et al., 2004). Arabidopsis seeds were surface sterilized and cold treated at 48C for 2 to 3 d and were then placed on solid 13 Murashige and Skoog medium supplemented with 1% sucrose for biochemical assays and phenotypical analysis. Cold-treated seeds were exposed to white light for 3 h and then transferred to continuous white light (30 mmol m 22 s 21 ) or continuous darkness, unless otherwise stated. Flowering times were tested in a chamber set for SDs (8 h light/16 h dark, 150 mmol m 22 s 21 ) or LDs (16 h light/8 h dark, 150 mmol m 22 s 21 ). Generation of Transgenic Arabidopsis Plants The full-length open reading frame of DDB1B was amplified by RT-PCR from wild-type Arabidopsis seedlings using the forward primer 59-TGGGGCCCGGAGGTGGCATGAGCGTATGGAACTACGCC-39 and the reverse primer 59-ATGGTCGACTCAGTGAAGCCTAGTGAGTTCTT- CAAC-39. The PCR product was cloned into the vector pcr2.1-topo (Invitrogen), and three copies of the Flag peptide were fused in-frame to the 59 end of DDB1B. Three groups of mutations within BPA, BPB, and BPC domains were generated separately. BamHI/SpeI fragments containing mutated flag-ddb1b were subcloned into Gateway vector pentr 1A (Invitrogen) and then recombined by the LR reaction into the plant binary vector pgwb2 that includes both kanamycin and hygromycin resistance markers and the 35S promoter of the Cauliflower mosaic virus. These constructs were then transformed into mutant ddb1a, and transgenic plants were selected with kanamycin (50 mg/ml; Sigma-Aldrich). The three groups of transgenic plants with mutated flag-ddb1b were named mbpa, mbpb, and mbpc, respectively. The anti-flag antibody (Sigma-Aldrich) was used to evaluate the level of fusion protein in total protein extracts. RT-PCR To analyze DDB1B transcript levels, total RNA was extracted from the plants using the Plant RNeasy Kit (Qiagen) according to the manufacturer s instruction. The concentration of RNA was determined by spectrophotometric measurements. The cdna was synthesized from 5 mg total RNA using oligo(dt)18 primer and superscript II reverse transcriptase enzyme (Invitrogen). DDB1B cdnas were amplified for 30 cycles at 948C for 45 s, 558C for 45 s, and 728C for 1 min. The forward and reverse primers for DDB1B were as follows: 59-GGGATAGGAATAAATGAA- CAG-39and 59-CTTCTGAATGTCATCGATGGTACC-39. To analyze CO, FT, and ACTIN2 transcript levels, total RNA was extracted from the green parts of soil-grown plants using the Plant RNeasy Kit according to the manufacturer s instructions. The concentration of RNA was determined spectrophotometrically. cdna was synthesized from 5 mg total RNA using oligo(dt)19 primer and superscript II reverse transcriptase (Invitrogen). cdnas were diluted to 200 ml with water, and 5 ml of the diluted cdna was used for PCR amplification. A CO fragment was amplified using primers CO53, 59-ACGCCATCAGCGAGTTCC-39, and COoli9, 59-AAATGTATGCGTTATGGTTAATGG-39 (Suarez-Lopez et al., 2001). An FT fragment was amplified using primers FT-RTPCR-F, 59-AGAAGAC- TTTAGATGGCTTCTT-39, and FT-RTPCR-R, 59-TTATCGCATCACACAC- TATATAAG-39 (An et al., 2004). Twenty-five and 27 cycles were used for CO and FT PCR, respectively. A fragment encoding ACTIN2 was amplified as an internal standard with primers described previously (Lee et al., 2008). The PCR products were analyzed by agarose gel electrophoresis using ethidium bromide staining. All RT-PCR analyses were repeated three times, and one representative result is shown. Quantitative Real-Time PCR To quantify FT and CO transcript levels in wild-type and cul4cs plants, the green parts of soil-grown plants were harvested for RNA extraction. Total RNA was extracted using the Plant RNeasy Kit. Reverse transcription was performed using TaqMan reverse transcription reagents (Applied Biosystems) according to the manufacturer s instructions. Quantitative PCR was performed using the Applied Biosystems 7900HT real-time PCR system with Taqman gene expression probes for FT, CO, and 18S rrna. Relative amounts of FT and CO transcripts were calculated using the comparative CT method, which was normalized against 18S rrna expression from the same sample. Each experiment was repeated twice with independent samples, and RT-PCR reactions were performed in duplicate for each sample. Antibodies and Immunoblot Assays The primary antibodies used in this study were anti-cul4 (Chen et al., 2006), anti-det1 and anti-ddb1 (Zhang et al., 2008), anti-cop1 (Saijo et al., 2003), anti-spa1 (Zhu et al., 2008), anti-flag, anti-myc, and anti- GST (Sigma-Aldrich), and anti-mbp (NEB). For the immunoblot analyses, Arabidopsis tissues were homogenized in an extraction buffer containing 50 mm Tris-HCl, ph 7.5, 150 mm NaCl, 10 mm MgCl 2, 0.1% Nonidet P-40, 1 mm phenylmethylsulfonyl fluoride, and 13 complete protease inhibitor (Roche). The extracts were centrifuged at 13,000g, and then protein concentrations in the supernatants were determined by the Bradford assay (Bio-Rad). Protein samples boiled in the sample buffer were run on SDS-PAGE, blotted onto polyvinylidene difluoride membranes (Millipore), and probed with different primary antibodies. Gel Filtration Chromatography For gel filtration analysis, 6-d-old Arabidopsis seedlings were homogenized in lysis buffer: 50 mm Tris-HCl, ph 7.5, 150 mm NaCl, 10 mm MgCl 2, 10 mm NaF, 2 mm Na 3 VO 4, 25 mm b-glycerophosphate, 10% glycerol, 0.1% Nonidet P-40, 1 mm phenylmethylsulfonyl fluoride, and 13 complete protease inhibitor cocktail (Roche). The extracts were centrifuged at 13,000g for 15 min at 48C and subsequently filtered through a 0.22-mm syringe filter. Superose 6 and superdex 200 columns (Amersham Biosciences) were used to fractionate the sample. After the void volume was eluted, consecutive fractions were collected, concentrated using Strataclean resin (Stratagene), and analyzed by protein gel blots. In Vitro Pull-Down Assay Arabidopsis DDB1A and DDB1B full-length coding sequences were subcloned into the pgex-4t-1 vector (Amersham). Arabidopsis full-length

14 CUL4-DDB1 Associates with COP1-SPA Complexes 121 SPA1, SPA2, SPA3, and SPA4 coding sequences were subcloned into pet28a (Novagen). MBP-COP1 and MBP-COP1mWDXR were purified as previously described (Saijo et al., 2003). MBP-COP1mWDXR was generated using the QuikChange site-directed mutagenesis kit (Stratagene). Full-length DDB1A and DDB1B fused to GST were expressed and purified from Escherichia coli strain RIL (DE3) (Stratagene) and induced by isopropyl-b-d-thiogalactopyranoside at 10 to 138C. GST protein was purified from E. coli BL21 (DE3) (Novagen) as a bait control. His- SPA1, His-SPA2, His-SPA3, His-SPA4, His-SPA1mWDXR, and His- SPA3mWDXR were synthesized by the TNT (Transcription/Translation) coupled in vitro reaction system (Promega). His-SPA1mWDXR and His- SPA3mWDXR were generated using the QuikChange site-directed mutagenesis kit. Bait and prey proteins were incubated in reaction buffer (50 mm Tris-HCl, ph 7.5, 150 mm NaCl, and 0.1% Nonidet P-40) for 4 h at 48C, and anti-gst conjugated beads were then added to precipitate the bait and bound prey proteins. Two micrograms of both bait and prey proteins (wild-type and mutated MBP-COP1) were used for each pulldown assay. The amounts of wild-type and mutated His-SPA proteins used were according to the specifications in the TNT system protocol (Promega). Immunoprecipitated proteins were resolved by SDS-PAGE and detected by Coomassie Brilliant Blue staining, immunoblot, and autoradiography. The quantification of the images was performed with ImageJ ( Relative band intensities were normalized with the value of background region and then calculated. In Vivo Coimmunoprecipitation Assay About 1 mg total proteins extracted from Arabidopsis tissues were incubated with antibody-conjugated agarose beads (Sigma-Aldrich) or IgG-agarose beads (Amersham) for 4 h at 48C. Alternatively, total proteins were incubated with anti-cop1 or anti-det1 antibodies for 3 h at 48C, and then protein A-agarose beads (Sigma-Aldrich) were added to each sample and incubated for 1 h at 48C. Lysis buffer was the same as used for gel filtration. Next, the beads were washed three times in the same buffer. Following elution, protein blot analyses were performed as described previously (Zhu et al., 2008). Accession Numbers Sequence data from this article can be found in the Arabidopsis Genome Initiative database under the following accession numbers: CUL4 (At5g46210), DDB1A (At4g05420), DDB1B (At4g21100), COP1 (At2g32950), SPA1 (At2g46340), SPA2 (At4g11110), SPA3 (At3g15354), SPA4 (At1g53090), ACTIN2 (At1g49240), CO (At5g15840), and FT (At1g65480). Supplemental Data The following materials are available in the online version of this article. Supplemental Figure 1. Mutated MBP-COP1, His-SPA1, and His- SPA3 Are Expressed at the Same Level as the Wild-Type Proteins. Supplemental Figure 2. DDB1 Associates with TAP-SPA1, TAP- SPA3, and TAP-SPA4 in Vivo. ACKNOWLEDGMENTS We thank Ning Zheng for helping with design for the point mutations on the surface of DDB1 protein, Tsuyoshi Nakagawa for providing the binary vector pgwb2 vector, Liying Chen for participation in some preliminary tests, Hui Cong and Hehe Chen for communication and support, and other members of the Deng lab for their constructive discussion and help. This research was supported by funds from National Institutes of Health (GM47850) and the National Science Foundation 2010 program and in part by the Ministry of Science and Technology of China and Beijing Commission of Science and Technology. H.C. was a Peking-Yale Joint Center Monsanto Fellow. Received January 6, 2009; revised November 2, 2009; accepted December 14, 2009; published January 8, REFERENCES Abbas, T., Sivaprasad, U., Terai, K., Amador, V., Pagano, M., and Dutta, A. (2008). 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17 Arabidopsis CULLIN4-Damaged DNA Binding Protein 1 Interacts with CONSTITUTIVELY PHOTOMORPHOGENIC1-SUPPRESSOR OF PHYA Complexes to Regulate Photomorphogenesis and Flowering Time Haodong Chen, Xi Huang, Giuliana Gusmaroli, William Terzaghi, On Sun Lau, Yuki Yanagawa, Yu Zhang, Jigang Li, Jae-Hoon Lee, Danmeng Zhu and Xing Wang Deng PLANT CELL 2010;22; ; originally published online Jan 8, 2010; DOI: /tpc This information is current as of March 3, 2010 Supplemental Data References Permissions etocs CiteTrack Alerts Subscription Information This article cites 62 articles, 35 of which you can access for free at: Sign up for etocs for THE PLANT CELL at: Sign up for CiteTrack Alerts for Plant Cell at: Subscription information for The Plant Cell and Plant Physiology is available at: American Society of Plant Biologists ADVANCING THE SCIENCE OF PLANT BIOLOGY

18 Supplemental Data. Chen et al. (2010). Plant Cell /tpc Supplemental Figure 1. Mutated MBP-COP1, His-SPA1, and His-SPA3 are Expressed at the Same Level as the WT Proteins. Recombinant MBP-COP1 and MBP-COP1mWDXR were purified from E.coli. 35 S-labeled His-SPA1, His- SPA1mWDXR, His-SPA3, and His-SPA3mWDXR were generated by the TNT system. The amount of proteins loaded here are 10% of the input proteins used for the in vitro pull down assay shown in Figure 2C. After separation by SDS-PAGE, proteins were visualized by anti-mbp immunoblot (for wild-type and mutated MBP-COP1), and autoradiography using X-ray films (for the SPA proteins). 1

19 Supplemental Data. Chen et al. (2010). Plant Cell /tpc Supplemental Figure2. DDB1 associates with TAP-SPA1, TAP-SPA3, and TAP- SPA4 in vivo. Total protein extracts prepared from six-day-old light-grown seedlings of wild-type, 35S:TAP-SPA1/spa1-3, 35S:TAP-SPA2/spa1-3 spa2-1, 35S:TAP-SPA3/spa3-1, and 35S:TAP-SPA4/spa4-1 transgenic Arabidopsis plants were incubated with IgGcoupled Sepharose. The precipitates and total extracts were subjected to immunoblot analysis with antibodies against Myc, COP1, and DDB1. Stars indicate the DDB1 protein band. 2